Various attempts for producing heterologous proteins in yeast using genetic engineering technology have been made. For example, HB vaccine for preventing Hepatitis B, which has been prepared by collecting and purifying the surface antigen protein of HB virus (HBV) from HBsAg positive human plasma and thus suffered shortage of supply and high cost, is recently produced by genetic engineering technology. More particularly, HBV genome DNA is cloned and gene coding for the surface antigen is isolated therefrom and the resulting HBsAg gene is expressed in yeast (K. Murray et al., EMBO J., 3:3,645 (1984), G. A. Bitter & K. M. Egan, Gene, 32:263 (1984)) or in animal cells (M. F. Dubois et al., Proc. Natl. Acad. Sci., 77, 4549 (1980)) to give HB vaccine in large amounts.
The human serum-derived HBsAg is in the form of 22 nm particles which are mainly composed of the peptide P24 (molecular weight 24,000) and the peptide P27 (molecular weight 27,000), which is glycosylated form of P24. In addition, they contain a minute amount of human serum-derived HBsAg particles peptide P31 (molecular weight 31,000), which is composed of P24 and 55 amino acid residues attached to the N-terminus of P24 (hereinafter referred to "3rd Pre S-HBsAg"), and the peptide P33 (molecular weight 33,000), which is glycosylated form of P31. Further, HBV contains as the surface antigen the peptide P41, which is composed of P31 and 108 or 119 amino acid residues attached to the N-terminus of P31 (hereinafter referred to "1st Pre S-HBsAg"), and the peptide P43, which is glycosylated form of P41.
The amino acid sequence of P31 contains attached thereto 55 amino acid residues encoded by the Pre S region present upstream from S antigen gene. The region composed of 55 amino acid residues is found to be capable of binding polymerized human serum albumin (pHSA) (Machida et al., Gastroenterol., 85:268 (1983)). HBV is believed to invade and infect human hepatocytes through pHSA. From this it follows that the antibody to the polyalbumin receptor is expected to play an important role in the prevention of infection of HBV. Since the polyalbumin receptor is encoded by the Pre S region of HB virus, HBsAg having the Pre S region gene product, i.e., Pre S-HBsAg, is believed to induce increased antibody production in humans as compared with HBsAg having no Pre S region polypeptide.
Peptide P31 composed of S antigen containing 55 amino acid sequence encoded by the Pre S region of HBV, i.e., 3rd Pre S-HBsAg, has been produced in large amounts by gene engineering technology using as a host yeast (P. Valenzuela et al., BIO/TECHNOLOGY, 3:317 (1985)) or animal cell (M. L. Michel et al., Proc. Natl. Sci., 81:7708 (1984)).
When the expression of genes coding for HBsAg by gene engineering technology is carried out HBsAg, 3rd Pre S-HBsAg and amino acid sequence composed of 3rd Pre S-HBsAg (2nd Pre S-HBsAg) and additional 35 amino acid residues attached to the N-terminus thereof can form particles in large amounts independently of others in contrast to 1st Pre S-HBsAg which is difficult to produce in large amounts by the expression of the gene coding therefor in yeast or animal cell independently of others.
On the other hand, when heterologous proteins are produced in yeast by gene engineering technology promoter regions of yeast origin, e.g., PH05 (K. Murray et al., EMBO J., 3 (3):645-650 (1984)), GAP-DH (G. A. Bitter & K. M. Eagan, Gene, 32:263-274 (1984)), PGK (C. Y. Chen et al., Nucl. Acid Res., 12 (23):8951-8970 (1984)), and .alpha.-Factor (A. J. Brake et al., Proc. Natl. Acad. Sci., U.S.A., 81:4642-4646 (1984)) are used. Generally, the promoter region in yeast in which RNA polymerase II acts is composed of two regions one containing the portion starting from the translation initiation site through the transcription initiation site and TATA box and another containing upstream activation site (UAS) which is present on the 5' side of the above-described region and functions in the cis mode to promote the transcription. (C. Guarente, Cell: 36, 799 (1984)). It has been shown that the UAS promotes the transcription of specific gene upon the action of a factor functioning in the trans mode in the cell (E. Giniger et al., Cell, 40:767-774 (1985)). Also, it has been shown that since the UAS functions independently of the region containing the sequence starting from the translation initiation site through the transcription initiation site the replacement of this UAS with the UAS of another gene leads to the release of the promoter from the original control system and the placement of the promoter under the control involving the new substitute UAS (L. Guarente et al., Proc. Natl. Acad. Sci., U.S.A., 79:7410 (1982); L. Guarente et al., Cell, 36:503 (1984); K. Struhl, Proc. Natl. Acad. Sci., U.S.A., 81:7865 ( 1984)).
The existence of enhancers which function in the cis mode to promote the transcription in the transcription-controlling region in the genes of animal cells and animal virus such as SV40 virus is shown (G. Khoury & P Gruss, Cell, 33:313-314 (1983)). Further, it has been shown that the enhancer of SV40 virus can function in the kidney cells of Green monkey (D. H. Hamer & P. Leder, Nature, 281:35 (1979)), COS cells (Humphries et al., Cell, 30:173 (1982)) and Hela cells (J. Banerji et al., Cell, 27:299 (1981)). Whether the enhancer of SV40 virus can function in yeast or not is unknown. However, it is interesting that it is shown that the Ty element of yeast contains a sequence homologous to the sequence of SV40 enhancer (B. Errede et al., Proc. Natl. Acad. Sci., U.S.A., 82:5423-5427 (1985)).